RECIPIENT: Lankenau Institute for Medical Research, Wynnewood, PA 19096
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1 AD AWARD NUMBER: W81XWH TITLE: Novel Therapeutic Target for the Treatment of Lupus PRINCIPAL INVESTIGATOR: Lisa Laury-Kleintop, PhD RECIPIENT: Lankenau Institute for Medical Research, Wynnewood, PA REPORT DATE: December 2013 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
2 REPORT DOCUMENTATION PAGE Form Approved OMB No Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports ( ), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE December 2013 Annual 3. DATES COVERED 01 July November TITLE AND SUBTITLE 5a. CONTRACT NUMBER Novel Therapeutic Target for the Treatment of Lupus 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Lisa Laury-Kleintop, Ph.D. 5e. TASK NUMBER 5f. WORK UNIT NUMBER KleintopL@mlhs.org 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER Lankenau Institute for Medical Research Wynnewood, PA SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Many therapeutic strategies for SLE focus on the central role that autoantibody-producing B cells play in the pathology of this disorder. One general immunotherapeutic theme employs monoclonal antibodies (mab) to interfere with, and/or deplete, B cells to ultimately reduce disease causing autoantibody levels. However, current strategies are inherently limited because they are not specific for the disease state. Thus, treatments that can specifically block autoantibody production without compromising B cell function are needed. In our application we presented preliminary evidence in an in vivo model of a related autoimmune disease (rheumatoid arthritis) that showed antibodies to RhoB, a small GTPase blocked autoantibody secretion without affecting the overall B cell repertoire. This data led us to propose the purpose of this study, to evaluate the ability of anti-rhob antibodies to reduce levels of pathologic autoantibodies, ultimately attenuating the severity of symptoms in the MRL-lpr murine model of SLE. We have not yet completed testing the anti-rhob therapy in the SLE model and subsequently received approval of an extension without funds for the award period. Thus far, our data suggests that dosing with the anti-rhob antibodies produces a trend towards a decrease in autoantibody titers and proteinuria; however, we are currently performing another treatment experiment that we hope will enable us to achieve statistical significance and have a better understanding of the effects resulting from targeting RhoB. 15. SUBJECT TERMS RhoB, animal model, antibody secretion, antibody therapy, Systemic lupus erythematosus, autoantibodies. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U 18. NUMBER OF PAGES c. THIS PAGE U UU 6 19a. NAME OF RESPONSIBLE PERSON USAMRMC 19b. TELEPHONE NUMBER (include area code)
3 Table of Contents Page 1. Introduction 2. Keywords 3. Overall Project Summary 4. Key Research Accomplishments 5. Conclusion 6. Publications, Abstracts, and Presentations 7. Inventions, Patents and Licenses 8. Reportable Outcomes 9. Other Achievements 10. References 11. Appendices
4 1. INTRODUCTION: Systemic lupus erythematosus (SLE) affects approximately 300,000 to over a million individuals in the United States with a female gender bias of 90%. Many therapeutic strategies for SLE focus on the central role that autoantibody-producing B cells play in the pathology of this disorder. One general immunotherapeutic theme employs monoclonal antibodies (mab) to interfere with and/or deplete B cells to ultimately reduce disease causing autoantibody levels. However, current strategies are inherently limited because they are not specific for the disease state. Thus, treatments that can specifically block autoantibody production without compromising B cell function remain lacking. Our work aims to address this therapeutic gap. The objective of our study is to evaluate the ability of anti- RhoB peptide antibodies to reduce levels of pathologic autoantibodies, ultimately attenuating the severity of symptoms in the MRL-lpr murine model of SLE. Our research strategy includes the determination of autoantibody levels over the course of disease progression in animals treated with therapeutic and control antibodies. Additionally, autoantibody levels will be compared to renal pathology to correlate autoantibody levels with attenuation of disease symptoms. This study will lay the groundwork for developing an innovative therapeutic strategy to improve the care of SLE patients, address important and timely scientific questions, as well as, focus on a major unmet medical need. 2. KEYWORDS: RhoB, animal model, antibody secretion, antibody therapy, Systemic lupus erythematosus, autoantibodies. 3. OVERALL PROJECT SUMMARY: Objective to complete in the award period of 18 months. Evaluate the ability of anti-rhob antibodies to attenuate the severity of symptom in the MRL/MpJ-Faslpr (abbrev. MRL-lpr) animal model of SLE. Task I. Prepare anti-rhob antibody. Months 1-3 Task II. Obtain approval for animal study. Months 1-3 During the first 3 months of the award period the first two tasks listed in the SOW were completed. During this time we refined the methodology and began purification of the RhoB antibodies (7F7 and 9G5), as well as, completed the approval process for our animal studies. Unfortunately, during this time it was determined that we had a mite outbreak in the animal facility and this outbreak has complicated the completion of the remaining tasks. Task III. Obtain MRL/MpJ-Faslpr female mice from approved vendor. Month 3 Task IV. Administer anti-rhob antibody to MRL-lpr mice and monitor for the development of autoantibodies and proteinuria. Mice will be dosed regularly during the course of the experiment, approximately 6 months. We anticipate repeating the experiment to reach statistical significance. Months 3-15 Task V. Perform ELISA analyses to evaluate serum autoantibody levels. Months 3-15 Task VI. Final serum autoantibody levels will be determined, as well as the levels of cells actively secreting anti-chromatin antibodies by ELISpot from isolated spleens. Renal function will be determined by monitoring for proteinuria. Renal pathology will be scored for nephritis using histological kidney sections. Months Task VII. Statistically analyze the data. Months10-18 We are currently working to complete tasks 3 through 7. Text included in the Progress Report submitted July Because of the detection of mites in the animal facility we did not import mice until the treatment for the outbreak was completed. All mice in the facility were treated topically with a 5% moxadectin solution (August 2012). We waited to import
5 mice and begin treatment until testing results indicated the mite outbreak was cleared (October 2012). However, approximately 3 weeks after we had imported and begun treating the first set of MRL-lpr mice with our targeted therapy, mites were again found in the facility. With this second outbreak all animals in the facility were fed ivermectin-containing food, and in our study, we continued to treat the MRL-lpr mice with our RhoB-targeted therapy. Analysis of the autoantibody levels from this group of mice did not show differences between the groups. However, we are concerned about the significance of the result from this first set of animals because ivermectin treatment has been shown to affect immune responses [1-3]. Additionally, in two other mouse models of autoimmune disease that received the ivermectin containing food, changes were observed in altered disease development and antibody levels as compared to studies done prior to the detection of mites and subsequent ivermectin treatment. In February 2013, we imported a second group of MRL-lpr mice after ivermectin treatment ended and sentinel screening was clear. The mice were divided into 3 treatment groups that received anti-rhob antibody 7F7 or 9G5, or mouse IgG as a control. Dosing began at 4 weeks of age and continued one dose per week until the end of the experiment. We assessed the development of serum autoantibody titers and proteinuria. The data presented in Figure 1, panel A suggest a trend towards a decrease in titers for anti-double stranded DNA antibodies in the groups treated with the anti-rhob antibodies compared to control IgG. Additionally, the data shown in Figure 1, panel B suggests a trending decrease in proteinuria with treatment of the anti-rhob antibodies. None of these differences reached statistical significance. Kidney tissue from these mice was isolated and histological samples will be scored to assess renal pathology. The data presented in Figure 1 suggests that anti-rhob antibodies may affect autoantibody titer and proteinuria in MRL-lpr mice; however, additional numbers are needed to achieve statistical significance. Figure 1: Autoantibody and proteinuria levels from MRL-lpr mice treated with anti-rhob antibodies 7F7 or 9G5. Panel A: ELISA results for serum antibody titers to double-stranded DNA. Serum titers were determined at indicated ages during the treatment period. Anti-RhoB antibodies 7F7 and 9G5 were compared to control mouse IgG. Panel B: Proteinuria was determined at 16 weeks of age prior to euthanasia. N=4-5 mice per group.
6 During the remaining period of the award we will repeat our analysis using additional mice. We are in the process of adding animal numbers to our IACUC and DOD animal protocols. Once appropriate approvals have been received we will continue with our study. We have two items to report since the submission of the Progress Report in July First, we have received approval of our amended animal protocol from both our IACUC and the DOD. Once approved, we purchased mice to continue our study. The mice were divided into a control group that is receiving control IgG or a treatment group receiving anti-rhob antibody 7F7. We chose to focus our attention on the effects of 7F7 because of new data regarding the antigen affinities of 7F7 and 9G5. Using recombinant proteins we characterized the cross-reactivity of the antibodies for other members of the small GTPase family. We found that 7F7 only recognized RhoB in elisa assays, while 9G5 recognized RhoB, RhoA and cdc42. Because 7F7 appears specific for RhoB, we plan to determine its therapeutic potential. It is still early in the treatment period and over the coming months we will perform assays to monitor autoantibody levels. Second, we had the kidney tissue from second cohort of mice (February 2013) scored to assess renal pathology. The results suggested that mice in the 9G5 treated group had a reduced degree of renal involvement exhibited by attenuated glomerular cellularity and basement membrane thickening. In light of our affinity data that 9G5 showed cross-reactivity with RhoA and cdc42, it may be that one or both of these other small GTPases play a role in the development of renal disease in this syndrome. We will have kidney tissue scored for renal disease from our current groups once treatment has been completed. 4. KEY RESEARCH ACCOMPLISHMENTS: There are no research accomplishments thus far from the study. 5. CONCLUSIONS: At present we are not able to conclude that our therapy can affect autoantibody levels in the MRL-lpr model of SLE. We will continue to work to meet the objective of our study, to evaluate the ability of anti-rhob peptide antibodies to reduce levels of pathologic autoantibodies, which we believe will attenuate the severity of symptoms in this animal model. Despite the setback of fur mites in the animal facility, we plan to meet our goal and obtain preliminary data necessary to advance an innovative therapeutic strategy into the clinic for treatment of SLE patients. 6. PUBLICATIONS, ABSTRACTS, AND PRESENTATIONS: None. 7. INVENTIONS, PATENTS AND LICENSES: None. 8. REPORTABLE OUTCOMES: There are no reportable outcomes thus far from the study. 9. OTHER ACHIEVEMENTS: None. 10. REFERENCES: 1. Blakley, B.R. and C.G. Rousseaux, Effect of ivermectin on the immune response in mice. Am J Vet Res, (4): p Yan, S., X. Ci, N. Chen, C. Chen, X. Li, X. Chu, J. Li, and X. Deng, Anti-inflammatory effects of ivermectin in mouse model of allergic asthma. Inflamm Res, (6): p Roble, G.S., W. Boteler, E. Riedel, and N.S. Lipman, Total IgE as a serodiagnostic marker to aid murine fur mite detection. J Am Assoc Lab Anim Sci, (2): p APPENDICES: None.
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AD Award Number: W81XWH-10-1-0450 TITLE: The role of NF1 in memory retrieval PRINCIPAL INVESTIGATOR: Yi Zhong, Ph.D. CONTRACTING ORGANIZATION: Cold Spring Harbor Laboratory Cold Spring Harbor, NY 11724
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AD AWARD NUMBER: W81XWH-05-1-0475 TITLE: Restoration of Epithelial Polarity in Metastatic Tumors PRINCIPAL INVESTIGATOR: Sergei Sokol, Ph.D. CONTRACTING ORGANIZATION: Mount Sinai School of Medicine New
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AD Award Number: W81XWH-06-1-0524 TITLE: Elucidating and Modeling Irradiation Effects on Centrosomal and Chromosomal Stability within Breast Cancer PRINCIPAL INVESTIGATOR: Christopher A. Maxwell, Ph.D.
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AD (Leave blank) Award Number: W81XWH-09-2-0065 TITLE: Trial of Naltrexone and Dextromethorphan for Gulf War Veterans Illnesses PRINCIPAL INVESTIGATOR: William J. Meggs, MD, PhD CONTRACTING ORGANIZATION:
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AWARD NUMBER: W81XWH-10-1-0711 TITLE: Transgenerational Radiation Epigenetics PRINCIPAL INVESTIGATOR: Christopher J. Kemp, Ph.D. CONTRACTING ORGANIZATION: Fred Hutchinson Cancer Research Center Seattle,
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AWARD NUMBER: W81XWH-13-1-0463 TITLE: The Ketogenic Diet and Potassium Channel Function PRINCIPAL INVESTIGATOR: Dr. Geoffrey Murphy CONTRACTING ORGANIZATION: University of Michigan Ann Arbor, MI 48109
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AD Award Number: W81XWH-04-1-0618 TITLE: Are Breast Tumor Stem Cells Responsible for Metastasis and Angiogenesis PRINCIPAL INVESTIGATOR: Quintin Pan, Ph.D. CONTRACTING ORGANIZATION: University of Michigan
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AWARD NUMBER: W81XWH-13-1-0479 TITLE: Sleep-Disordered Breathing in Chronic SCI: A Randomized Controlled Trial of Treatment Impact on Cognition, Quality of Life, and Cardiovascular Disease PRINCIPAL INVESTIGATOR:
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AD Award Number: W81XWH-12-1-0212 TITLE: Wnt/Beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression PRINCIPAL INVESTIGATOR: Xiuping Yu CONTRACTING ORGANIZATION: Vanderbilt University
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AD Award Number: W81XWH-11-1-0126 TITLE: Chemical strategy to translate genetic/epigenetic mechanisms to breast cancer therapeutics PRINCIPAL INVESTIGATOR: Xiang-Dong Fu, PhD CONTRACTING ORGANIZATION:
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AD Award Number: W81XWH-11-1-0604 TITLE: An Intervention to Control Vasomotor Symptoms for Advanced PC Patients on Hormone Therapy PRINCIPAL INVESTIGATOR: Michael A. Diefenbach, Ph.D. CONTRACTING ORGANIZATION:
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AD Award Number: W81XWH-08-2-0050 TITLE: PT073853: Mild TBI Following Exposure to Explosive Devices: Device Characteristics, Neuropsychological Functioning, and Symptoms of Post-Traumatic Stress Disorder
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More informationApproved for public release; distribution unlimited
Award Number: W81XWH-10-1-1021 TITLE: Post-traumatic Headache and Psychological Health: Mindfulness Training for Mild Traumatic Brain Injury PRINCIPAL INVESTIGATOR: Sutapa Ford, PhD CONTRACTING ORGANIZATION:
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AD Award Number: W81XWH-12-1-0225 TITLE: Epstein Barr virus and blood brain barrier in Multiple Sclerosis PRINCIPAL INVESTIGATOR: Luwen Zhang CONTRACTING ORGANIZATION: University of Nebraska Lincoln, NE
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